Thienoquinolines as Novel Disruptors of the PKCε/RACK2 Protein–Protein Interaction

Florian Rechfeld,Nina Hauser,Markus Boehler,Johannes Kirchmair,Johann Hofmann,Gennady B Lapa,Dorota Garczarczyk,Peter Goekjian,Maria N Preobrazhenskaya,Peter Gruber,Georg Hechenberger,Thierry Langer

doi:10.1021/jm401605c

Abstract

Ten protein kinase C (PKC) isozymes play divergent roles in signal transduction. Because of sequence similarities, it is particularly difficult to generate isozyme-selective small molecule inhibitors. In order to identify such a selective binder, we derived a pharmacophore model from the peptide EAVSLKPT, a fragment of PKCε that inhibits the interaction of PKCε and receptor for activated C-kinase 2 (RACK2). A database of 330 000 molecules was screened in silico, leading to the discovery of a series of thienoquinolines that disrupt the interaction of PKCε with RACK2 in vitro. The most active molecule, N-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide (8), inhibited this interaction with a measured IC50 of 5.9 μM and the phosphorylation of downstream target Elk-1 in HeLa cells with an IC50 of 11.2 μM. Compound 8 interfered with MARCKS phosphorylation and TPA-induced translocation of PKCε (but not that of PKCδ) from the cytosol to the membrane. The compound reduced the migration of HeLa cells into a gap, reduced invasion through a reconstituted basement membrane matrix, and inhibited angiogenesis in a chicken egg assay.

Highlights

Protein kinase C (PKC) is a family of serine/threonine-specific protein kinases
A database of 330 000 molecules was screened in silico, leading to the discovery of a series of thienoquinolines that disrupt the interaction of PKCε with RACK2 in vitro
A crystal structure is only available for the N-terminal-C2-like domain of PKCε (PDB entry 1GMI), but no structural data are available on RACK2

Summary

Introduction

Protein kinase C (PKC) is a family of serine/threonine-specific protein kinases. The PKC isozymes can be classified into three groups: (i) the conventional α, βI, βII, γ; (ii) the novel δ, ε, θ, η, and (iii) the atypical λ/ι (mouse/human) and ζ. The PKCε isozyme has been reported to participate in neoplastic transformation,[1] cardiac hypertrophy,[2] protection from ischemic insult,[2,3] nociceptor function,[4] macrophage activation,[5] diabetes,[6] and alcohol consumption.[7] A PKCε isozyme-specific inhibitor would be a valuable tool for analyzing the function of PKCε and is expected to have pharmaceutical potential for cancer, stroke, drug addiction, or pain.[8,9] Typically, kinase inhibitors interact with the ATP-binding site, which is well conserved among different kinase families and is even more so within isoforms of a given kinase This poses a serious hurdle for the development of isozyme-specific inhibitors, as there are about 500 kinases encoded by the human genome.[10] several selective kinase inhibitors have been reported, it was later found that they inhibit other targets. Relatively unspecific inhibitors initially intended as PKCβ-selective, such as ruboxistaurine[11,12] and enzastaurine,[13] are in clinical trials for Received: October 16, 2013 Published: April 8, 2014

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